Overall Project Abstract Anemia is a major health problem affecting millions of individuals around the world. The overall objective of the proposed program is to develop an improved mechanistic understanding of erythropoiesis, with the goal of defining pathophysiological mechanisms resulting in anemia due to ineffective erythropoiesis. Our overarching hypothesis is that normal human erythropoiesis requires major changes in patterns of gene expression that impact key interconnected pathways in mitosis/cytokinesis, apoptosis, metabolite transport and nuclear structure. Comparisons of these pathways will be performed in a number of important red cell disorders. Four independent but complementary projects that rely on each other have been assembled to explore different aspects of human erythropoiesis. Project 1 will develop a comprehensive mechanistic understanding of the role of changes in gene expression in generating distinct erythroid populations with particular focus on the regulation of erythroid progenitors and late stages of terminal erythroid differentiation including enucleation in normal and disordered erythropoiesis. Project 2 will develop a mechanistic understanding of how the ordered synthesis of distinct proteins is regulated during erythropoiesis by identifying and characterizing enhancers regulating stage-specific programs of gene expression in highly specialized human erythroid cells. In parallel, proteins undergoing mRNA translation at different stages of erythroid development and differentiation will be defined and characterized by ribosomal profiling. Project 3 will develop a mechanistic understanding of the role of cell metabolism in erythroid differentiation. The role of cytokines in erythroid differentiation has been extensively studied but it is only recently that we have begun to recognize the importance of nutrient entry and metabolism in transitioning to different erythroid stages and the proposed project will use a novel scaffold of ligands to metabolite transporters in order to extend our fundamental knowledge of metabolism in erythroid differentiation. These data will reveal novel pathways by which metabolites regulate erythroid lineage differentiation and will result in the identification, and potential manipulation of nutrient transporters that orient erythroid progenitor survival and differentiation in physiological erythropoiesis as well as in the disordered erythropoiesis. Project 4 will develop mechanistic understanding of the role of cell and nuclear stiffness and polarized contractile forces in enucleation, marrow egress, and `self' recognition by macrophages of erythroid cells. The role of lamins, the main nuclear structural proteins in cells, in regulating nuclear rigidity and of myosin-driven cell polarization in these processes will be explored. These insights will result in improved mechanistic understanding of key cellular event during erythroid differentiation.